Osteoclasts are large, multinucleated cells that play a central role in bone resorption. They are derived from hematopoietic precursors in response to a number of regulatory factors, the most important of which is RANKL. RANKL is both necessary and sufficient for osteoclastogenesis, although factors such as M-CSF, TGFb, inflammatory cytokines and prostaglandins are also important in the events associated with this process. RANKL is synthesized in a number of cell types, including those of the osteoblast lineage. Studies both in vitro and in vivo indicate that RANKL mRNA expression is regulated physiologically by key bone remodeling hormones such as PTH and 1,25(OH)2D3 as well as by cytokines such as IL-1, TNFa, IL-6 and the prostaglandin PGE2. Aberrant production and/or expression of any one these modulators has been implicated in RANKL overexpression, enhanced bone resorption and osteolytic or osteoporotic disease. Despite considerable physiologic and pathologic insight, little is known of the molecular mechanisms that control the transcriptional output of RANKL from support cells. Initial studies, however, suggest that the mechanisms responsible are likely to be highly complex. In view of the critical impact of RANKL on bone resorption, we propose to define key mechanisms instrumental to RANKL gene expression at the molecular level.
Aim 1 : To define the molecular events associated with activation of the RANKL gene by VDR, CREB, and STATS at the RANKL distal control region (RL- DCR) located 76 kb upstream of the mouse RankL transcriptional start site (TSS). The relevance of this site will be confirmed using a genetically altered mouse model.
Aim 2 : To evaluate the contribution of four additional regulatory domains located upstream of the mouse RankL gene TSS at -16, -22, -60 and -69 kb. The contribution of these sites to the regulation of RankL gene expression will be examined through preparation of a unique mouse model.
Aim 3 : To assess the consequence of transcription factor occupancy within the RankL gene upstream region on chromatin organization, modification and structure, and to determine the function of RNA polymerase II which appears specifically at these sites in response to activation. These studies will provide important detail into the mechanism by which calciotropic hormones and cytokines regulate RANKL expression, a gene whose product is central to bone resorption.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK074993-05
Application #
8012870
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Malozowski, Saul N
Project Start
2007-01-15
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
5
Fiscal Year
2011
Total Cost
$286,484
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Onal, Melda; St John, Hillary C; Danielson, Allison L et al. (2016) Deletion of the Distal Tnfsf11 RL-D2 Enhancer That Contributes to PTH-Mediated RANKL Expression in Osteoblast Lineage Cells Results in a High Bone Mass Phenotype in Mice. J Bone Miner Res 31:416-29
Onal, M; St John, H C; Danielson, A L et al. (2016) Unique Distal Enhancers Linked to the Mouse Tnfsf11 Gene Direct Tissue-Specific and Inflammation-Induced Expression of RANKL. Endocrinology 157:482-96
Pike, J Wesley; Meyer, Mark B; Benkusky, Nancy A et al. (2016) Genomic Determinants of Vitamin D-Regulated Gene Expression. Vitam Horm 100:21-44
Pike, J Wesley; Meyer, Mark B; St John, Hillary C et al. (2015) Epigenetic histone modifications and master regulators as determinants of context dependent nuclear receptor activity in bone cells. Bone 81:757-64
Onal, Melda; Bishop, Kathleen A; St John, Hillary C et al. (2015) A DNA segment spanning the mouse Tnfsf11 transcription unit and its upstream regulatory domain rescues the pleiotropic biologic phenotype of the RANKL null mouse. J Bone Miner Res 30:855-68
Bishop, Kathleen A; Wang, Xiaohua; Coy, Heidi M et al. (2015) Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers. J Cell Biochem 116:320-30
Meyer, Mark B; Benkusky, Nancy A; Pike, J Wesley (2015) Profiling histone modifications by chromatin immunoprecipitation coupled to deep sequencing in skeletal cells. Methods Mol Biol 1226:61-70
Pike, J Wesley; Meyer, Mark B (2014) Fundamentals of vitamin D hormone-regulated gene expression. J Steroid Biochem Mol Biol 144 Pt A:5-11
Pike, J Wesley; Lee, Seong Min; Meyer, Mark B (2014) Regulation of gene expression by 1,25-dihydroxyvitamin D3 in bone cells: exploiting new approaches and defining new mechanisms. Bonekey Rep 3:482
Obr, Alison E; Grimm, Sandra L; Bishop, Kathleen A et al. (2013) Progesterone receptor and Stat5 signaling cross talk through RANKL in mammary epithelial cells. Mol Endocrinol 27:1808-24

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